Human umbilical cord mesenchymal stem cell-derived exosomes promote osteogenesis in glucocorticoid-induced osteoporosis through PI3K/AKT signaling pathway-mediated ferroptosis inhibition

Abstract

Glucocorticoid-induced osteoporosis (GIOP), the most common cause of secondary osteoporosis, is characterized by significant bone loss, decreased bone quality, and increased fracture risk. The current treatments for GIOP have several drawbacks. Exosomes are vital for cellular processes. However, very few studies have focused on using human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) for GIOP treatment. In vitro and in vivo dexamethasone was used to evaluate the therapeutic effects of hUCMSC-EXOs on GIOP. CCK-8 and EdU assays were used to evaluate cell viability and proliferation, respectively. We conducted an alkaline phosphatase activity assay, alizarin red staining, Western blotting, and real-time PCR to detect the effect on osteogenesis. TMT-labeled quantitative proteomic and bioinformatic analyses were performed. Furthermore, we performed Western blotting, immunofluorescence, reactive oxygen species assays, and lipid peroxidation assays to investigate the regulatory mechanism by which hUCMSC-EXOs affect cell proliferation and osteogenic differentiation. The in vivo effects of hUCMSC-EXOs were evaluated using micro-CT, hematoxylin, and eosin staining, and immunohistochemical staining. We found that hUCMSC-EXOs reversed the inhibitory effects of glucocorticoids on human bone marrow stromal cell (hBMSC) proliferation and osteogenic differentiation and demonstrated that hUCMSC-EXOs reversed GIOP via the PI3K/AKT signaling pathway, inhibiting lipid peroxidation in vitro and in vivo. HUCMSC-EXOs promote hBMSC osteogenesis through the PI3K/AKT signaling pathway, inhibit ferroptosis, and have therapeutic potential for GIOP in mice.

Keywords: PI3K/AKT signaling; exosome; ferroptosis; human bone marrow stromal cells; human umbilical cord mesenchymal stem cells; osteogenesis.

Graphical Abstract

Figure 1.

HUCMSC-EXOs counteracts the decreased proliferation induced by dexamethasone in hBMSCs in vitro. (A) HUCMSC-EXO morphology identification using transmission electron microscopy (TEM; scale bar100 nm). (B) HUCMSC-EXO particle size and concentration measurement using nanoparticle tracking analysis (NTA). (C) Western blotting assay of the surface biomarkers CD9, CD63, and TSG101. (D) HUCMSC-EXOs were labeled with PKH26 and hUCMSC-EXO endocytosis detection using an inverted fluorescence microscope. (E) HBMSC proliferation detection using CCK-8 assay. (F) The quantitative calculation of HBMSC proliferation. (G) DNA replication monitoring in hBMSCs using 5-ethynyl-2ʹ-deoxyuridine (EdU) staining (scale bar 100 μm). (H) EdU-positive percentage calculated using ImageJ.

Figure 2.

Effects of hUCMSC-EXOs on osteogenic differentiation of GC-treated hBMSCs in vitro. Western blotting, alkaline phosphatase (ALP) activity assay, and alizarin red staining (ARS) were used to evaluate the osteogenic differentiation of hBMSCs after different treatments. (A) After osteogenic differentiation induction, the effect of hUCMSC-EXOs on Col1a1, BMP2, OPN, and Gpx4 expression levels were determined by Western blotting. (B-E) Quantitative analysis of Western blots using ImageJ software. (F) ALP activity assay (scale bar 500 μm). (G) Relative ALP-positive area was calculated using ImageJ software. (H) ARS (scale bar 500 μm). (I) Relative Alizarin red-positive areas were calculated using ImageJ software.

Figure 3.

Functional attributes related to hUCMSC-EXO-induced changes in proteomic and signaling pathways. Proteomic comparison between the cells treated with dexamethasone in the presence or absence of hUCMSC-EXOs (Dex + EXOs and Dex groups). (A) Volcano plot showing differentially expressed protein, red upregulated, and blue downregulated. (B) Gene ontology (GO) biological process classification of genes. (C and D) KEGG functional enrichment analysis.

Figure 4.

HUCMSC-EXOs promote GC-treated hBMSC osteogenesis by activating PI3K/AKT signaling pathway in vitro. (A and B) Identification of the underlying correlation between PI3K/AKT signaling pathway and osteogenic differentiation using western blotting. (C–K) Quantitative analysis of Western blotting images using ImageJ. (L) Alkaline phosphatase (ALP) activity assay staining (scale bar 500 μm). (M) Relative ALP-positive area as calculated by ImageJ. (N) Alizarin red staining (scale bar 500 μm). (O) Relative Alizarin red-positive area as calculated by ImageJ. (P) p-AKT (Ser 473) expression detection using immunofluorescence. (Q) Quantitative analysis of immunofluorescence by ImageJ.

Figure 5.

HUCMSC-EXOs inhibit GC-treated hBMSC ferroptosis via PI3K/AKT signaling pathway in vitro. (A) Detection of DNA replication of hBMSCs after different treatments using 5-ethynyl-2ʹ-deoxyuridine (EdU) staining (scale bar 100 μm). (B) Quantitative analysis of EdU by ImageJ. (C) Gpx4 expression detection using immunofluorescence (scar bar 25 µm). (D) Quantitative analysis of immunofluorescence by ImageJ. (E) Western blotting was performed to evaluate Gpx4 expression with different interventions. (F) Quantitative analysis of Western blotting using ImageJ. (G) Oxidative stress level monitoring using reactive oxygen species (ROS) staining (scale bar 100 μm). (H) Quantitative analysis of the number of ROS-positive cells per field. (I) Detection of lipid peroxidation using the C11 BODIPY™ 581/591 fluorescent probe (scale bar 100 μm). (J) Quantitative analysis of oxidized/non-oxidized C11 ratio using ImageJ.

Figure 6.

HUCMSC-EXOs promoted GC-treated hBMSC osteogenesis by activating PI3K/AKT pathway-mediated ferroptosis inhibition in vitro. (A) Col1a1, BMP2, and OPN expression evaluation after different interventions using Western blotting. (B–D) Quantitative analysis of western blotting using ImageJ. (E–G) BMP2, ALP, and Runx2 expression levels in different groups were verified using real-time PCR. (H) Alkaline phosphatase (ALP) activity assay staining (scale bar 500 μm). (I) Relative ALP-positive area as calculated by ImageJ. (J) Alizarin red staining (scale bar 500 μm). (K) Relative Alizarin red-positive area as calculated by ImageJ.

Figure 7.

In vivo osteogenesis was regulated through PI3K/AKT pathway-mediated ferroptosis inhibition. (A) Micro-CT scan showing the trabecular levels of tibial plateau after different interventions and rectangle showing the region of interest. (B-E) Quantitative bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) analysis after different treatments. (F) Hematoxylin and eosin (HE) staining showing the distribution and morphology of bone trabeculae in bone tissue (scar bar 500 µm). (G) Immunohistochemical staining to detect p-AKT (Thr 308) expression levels in the femoral head of mice after different treatments (scar bar 100 µm). (H) Immunohistochemical staining to detect GPX4 expression levels in the tibial plateau of mice after different treatments (scar bar 100 µm).